Additive and/or synergistic combinations of metformin with nutraceuticals for the prevention, inhibition and treatment of sars-cov-2 and associated covid-19

ABSTRACT

Disclosed are compositions of matter, treatments and protocols useful for prevention of SARS-CoV-2 infection, as well as inhibition of viral propagation and acceleration of viral cure. In some embodiments the invention teaches the administration of a therapeutic combination of ingredients comprising of metformin, pterostilbene, nigella sativa, sulforaphane, and epigallocatechin-3-gallate (EGCG) to a mammal at risk of infection with SARS-CoV-2. In another embodiment, the invention teaches administration of said therapeutic combination to a mammal infected with said SARS-CoV-2. In some embodiments dosage of said therapeutic combination is based on inflammatory and/or immunological parameters observed in patients with COVID-19.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S. Provisional Application No. 63/054,805, filed Jul. 22, 2020, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the field of viral infections, more specifically, the invention pertains to treatment of viral infections using nutraceutical interventions.

BACKGROUND

SARS-CoV-2, the viral pathogen causative of COVID-19, is a novel coronavirus that is most phylogenetically similar to SARS. The virus is presumed to have initially been transmitted from an animal reservoir (bats) to humans, most likely via an amplifying host (pangolin) [1, 2]. It is a single strand positive sense RNA virus whose infectivity is mediated by the envelope spike (S) glycoprotein which binds to its cellular receptor angiotensin-converting enzyme 2 (ACE2) [3]. Interestingly, antibody responses have also been detected towards the S glycoprotein [3-6].

It is known that Coronaviruses (CoVs) belong to a subfamily of large and enveloped viruses containing a single strand of sense RNA. There are four genera, of CoVs, i.e., alpha, beta, gamma, and delta, of which alpha- and beta-CoVs are known to infect humans [7]. Infectivity of CoVs is mediated by the envelope spike (S) glycoprotein which binds to its cellular receptors angiotensin-converting enzyme 2 (ACE2) and dipeptidyl peptidase 4 (DPP4) for SARS-CoV and MERS-CoV, respectively, this facilitates fusion of the virus with the membrane [8, 9]. The viral RNA genome is released into the cytoplasm; after replication ofthe viral genome, genomic RNA accompanied by envelope glycoproteins and nucleocapsid proteins formsvirion-containing vesicles, which then fuse with the plasma membrane to release the virus [10]. The viral cause of COVID-19 is attributed to the SARS-CoV-2 type of CoV, which was found to be a new type of beta-CoV with more than 99.98% genetic identity among 10 sequenced samples collected from the originalsite of the outbreak [11]. SARS-CoV-2 is genetically more similar to SARS-CoV than to MERS-CoV [12].

Subsequent to the virus binding to the target cells, which appear to be primarily type 2 pulmonary epithelial cells, the virus fuses with the cellular membrane, allowing viral entry into the cytoplasm [13]. Once the viral genome is unleashed in the cell, it replicates and the viral RNA accompanied by envelope glycoproteins and nucleocapsid proteins form virion-containing vesicles, which then fuse with the plasma membrane to release the virus [14].

At a clinical level, the usual presentation of COVID-19 is pneumonia, as demonstrated by computer tomographic (CT) scan or chest X-ray and fever. At the beginning phases of the infection, the patients showed the acute respiratory infection symptoms, with some that quickly developed acute respiratory failure (ARDS) and other serious complications [15]. The original patients that were published from the China Novel Coronavirus Investigating and Research Team all developed severe pneumonia and two of these three patients with available clinical profiles showed a common feature of fever and cough [13].

After this, another study of a family of six patients in the University of Hong Kong-Shenzhen Hospital demonstrated that all of them had pulmonary infiltrates, and other flu-like characteristics [16]. The chest X-ray and CT imaging in a study showed that 75% of 99 patients possessed bilateral pneumonia and the remaining 25% unilateral pneumonia [17]. In total about, 14% of the patients showed multiple mottling and ground-glass opacity [17]. The first cases of coronavirus infection in the United States also showed basilar streaky opacities in both lungs by chest radiography. However, the pneumonia for this patient was only detected on the day 10 of his illness [18].

As reviewed by Zheng [1], identifiable characteristics of COVID19 patients are fever and cough, with fatigue in 96% of patients (n=138) in one study, but was less outstanding (18%, n=44) in another report. Overall analysis revealed that fever was observed in around 90% whereas cough is relatively less at 68%,

and shortness of breath, muscle ache, headache, chest pain, diarrhea, haemoptysis, sputum production, rhinorrhoea, nausea and vomiting, sore throat, confusion, and anorexia were also observed in an numerous patients.

Currently no interventions have demonstrated efficacy in double blind trials, however multiple approaches are in development. These can be categorized into a) antigen specific vaccines [19-22]; b) innate immune stimulators [23-25]; c) small molecule antivirals; d) small molecules which modulate viral interactions with host cells [26-29]; e) plasma/antibodies from patients who recovered [30-40]; f) small molecule blockers of cytokine signaling [41-44]; g) antibodies to inflammatory cytokines [45-53]; and h) cell based therapies [54-64].

At present there appears to be no clear consensus on which approaches are most promising, with various institutions utilizing differing protocols. It is expected that clinically significant signals will be reported inup to 18 months from now. In the absence of established pharmaceutical approaches, exploration of scientific based natural based treatments may have merit.

SUMMARY OF THE INVENTION

Preferred embodiments are directed to methods of inhibiting infection with SARS-CoV-2, suppressing proliferation of SARS-CoV-2 and inducing resolution of SARS-CoV-2 comprising administration of a therapeutic combination comprisingof: a) Green Tea and/or extract thereof; b) Blueberry and/or extract thereof; c) Nigella Sativa and/or extract thereof; d) broccoli and/or extract thereof and e) metformin

Preferred embodiments include methods wherein said green tea extract is epigallocatechin-3-gallate or an analoguethereof.

Preferred embodiments include methods wherein said blueberry extract is pterostilebene or an analogue thereof.

Preferred embodiments include methods wherein said Nigella Sativa extract is thymoquinone or an analogue thereof.

Preferred embodiments include methods wherein said broccoli extract is sulforaphane or an analogue thereof.

Preferred embodiments include methods wherein said therapeutic combination is administered at a dosage and frequency sufficient to inhibit viral establishment into the host.

Preferred embodiments include methods wherein inhibition of viral establishment into the host is accomplished byenhancement of natural killer cell activity.

Preferred embodiments include methods wherein said natural killer cell activity is quantified by ability to lyse a virallyinfected cell.

Preferred embodiments include methods wherein said natural killer cell activity is quantified by ability to lyse K562cells.

Preferred embodiments include methods wherein said natural killer cell activity is quantified by ability to lyse YAC-lcells.

Preferred embodiments include methods wherein inhibition of viral establishment into the host is accomplished byenhancement of interferon production.

Preferred embodiments include methods wherein inhibition of viral establishment into the host is accomplished byenhancement of T cell activation.

Preferred embodiments include methods wherein said T cell activation is induction of T helper cell 1 activity.

Preferred embodiments include methods wherein said T helper cell 1 activity comprises production of interferongamma.

Preferred embodiments include methods wherein said T cell activation is induction of T cytotoxic cell activity.

Preferred embodiments include methods wherein said therapeutic combination is administered at a dosage and frequency sufficient to inhibit viral replication into the host.

Preferred embodiments include methods wherein said viral replication in the host is associated with suppression ofthe viral life cycle.

Preferred embodiments include methods wherein said viral life cycle comprises of: a) entry; b) propagation; and c)budding.

Preferred embodiments include methods wherein said therapeutic mixture decreases propensity towards acuterespiratory distress syndrome (ARDS).

Preferred embodiments include methods wherein said ARDS is associated with activation of complement.

Preferred embodiments include methods wherein said complement activation is associated with C5a upregulation.

Preferred embodiments include methods wherein said ARDS is associated with mast cell degranulation.

Preferred embodiments include methods wherein said ARDS is associated with enhanced expression of adhesionmolecules on endothelial cells.

Preferred embodiments include methods wherein said adhesion molecules are selected from a group comprising of:

a) E selectin; b) ICAM-1; c) VLA-4 and; d) Cadherin.

Preferred embodiments include methods wherein said ARDS is associated with enhanced monocytic accumulation inthe alveolar space.

Preferred embodiments include methods wherein said monocytes are of the M1 lineage.

Preferred embodiments include methods wherein said monocytes are of the M2 lineage.

Preferred embodiments include methods wherein said ARDS is associated with enhanced neutrophil accumulation inthe alveolar space.

Preferred embodiments include methods wherein said neutrophils are activated.

Preferred embodiments include methods wherein said activated neutrophils produce matrix metalloproteases.

Preferred embodiments include methods wherein said therapeutic composition reduces expression of inflammatorymarkers.

Preferred embodiments include methods wherein said inflammatory markers are selected from a group comprisingof: Cluster of differentiation 40 ligand (CD-40L), Eotaxin, fibrinogen, growth hormone (GH), keratinocyte-derived cytokine (KC/GRO), interleukin-1.beta. (IL-1.beta.), IL-6, IL-18, lymphotactin, myeloperoxidase (MPO), tissue inhibitor of metalloproteinase 1 (TIMP-1), C-reactive protein (CRP), macrophage-derived chemokine (MDC), macrophage inflammatory protein-1.alpha. (MIP-1.alpha.), vWF, and oncostatin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing QUADRAMUNE™ and metformin reduce lung injury.

FIG. 2 is a bar graph showing QUADRAMUNE™ and metformin enhances Type 2 monocytes.

FIG. 3 is a bar graph showing QUADRAMUNE™ and metformin enhance anti-inflammatory protein IL-10.

FIG. 4 is a bar graph showing QUADRAMUNE™ and metformin reduce inflammatory protein IL-17.

FIG. 5 is a bar graph showing QUADRAMUNE™ and metformin enhance regenerative protein HGF-1.

DESCRIPTION OF THE INVENTION

The formulation of QuadraMune was based on the desire to develop a multi-angled nutraceutical solution which addressed various aspects of COVID-19. For example, in the beginning of the disease process, the body's susceptibility to infection with the SARS-CoV-2 virus is a major determining factor as to progression of disease. Belonging to the coronavirus family of viruses, it is well recognized that naturalkiller cells, and interferon responses play a role in host susceptibility. For example, in an early mouse studies it was demonstrated that interferon response negatively correlated with viral susceptibility [65, 66]. In other murine studies it has been demonstrated that successful infection with coronavirus involves viral suppression of the natural kill cell response [67]. Interestingly, agents which increase NK activity has been shown to decrease coronavirus infections. For example, in one study, a novel CpG oligonucleotide (BW001) was assessed, which displays B-type CpG ODN structure feature at the 5′ and A-type CpG ODN structure feature at the 3′, and tested for its anti-SARS-CoV activity. We found that the supernatants of human PBMCs stimulated by BW001 significantly protected Vero cells from SARS-CoV infection. BW001 could stimulate human PBMCs and pDCs to secrete high level of IFN-alpha and promote human PBMCs and B cells to proliferate. Furthermore, we demonstrated that BW001 could activate CD19+ B cells and CD56+ NK cells in human PBMCs. In addition, BW001 could enhance NK cytotoxicity and IFN-gamma secretion in human PBMCs [68].

Therefore, while there is rationale for stimulation of immunity at the beginning phases of infection, as infection progresses, various types of immunity may be detrimental. For example, it is known that pulmonary inflammation and progression of acute respiratory distress syndrome (ARDS) occurs as a result of excessive immune response. Mortality from COVID-19 is caused by acute respiratory distress syndrome (ARDS) [69, 70], which is caused by unrestrained cytokine release, also known as “cytokine storm”, and is characterized by fluid leakage, diffuse inflammation, and disseminated intravascular coagulation, all of which cause impaired alveolar gas exchange. Approximately 35-45% of patients with ARDS will die [71].

The role of inflammatory cytokines in the progression of ARDS and its pathology may be seen in several situations. For example, tumor necrosis factor (TNF)-alpha, has been demonstrated to correlate with severity of ARDS in several studies. In one study, measure plasma TNF alpha levels (pl-TNF alpha) in 34 patients with ARDS and in 16 controls was examined. Plasma, TNF alpha was elevated in 76% of the patients with ARDS (71+/−104 pg/ml) and in 48% of the at-risk patients (47+/−73 pg/ml), providing someindication that TNF-alpha may correlated with ARDS [72]. In another study assessment of TNF-alpha was performed in fourteen hospitalized patients with a diagnosis of SARS-associated coronavirus infection. All patients had fever, dry cough and dyspnea. Twelve were intubated during hospitalization. The median duration from onset of fever to the nadir level or most severe condition was 9 days for hypoxia. The 8 patients who died possessed significantly higher peak levels of serum TNF-alpha compared to those who survived (14 vs 9.1 pg/mL; p=0.06) [73]. Another study demonstrated correlation between TNF-alpha and mortality. The study examined ICU patients on ventilator with (n=9) and without (n=12) evidence of ARDS. The median peak TNF concentration in control patients was 40 ng/L (range less than 40-100 ng/L) and in ARDS patients 231 ng/L (range 100-2550 ng/L; p less than 0.001). All of the control patientswere discharged alive from the ICU, whereas 6 of 9 ARDS patients died in the ICU. In 6 ARDS patients, it was possible to measure more than 4 consecutive plasma TNF levels. Of these 6 patients, the 3 with persistent elevations in systemic TNF above 230 ng/L succumbed (p less than 0.05, one-tailed) [74].

It is believed the TNF-alpha production causes pathology in ARDS at several levels. In one experiment, TNF-alpha was administered intratracheally at 500 ng in healthy rats. It was observed that within 5 hours, lung lavage neutrophils, lung myeloperoxidase (MPO) activity, and lung leak was substantially higher in the treated as compared to saline-treated control rats [75]. In another study, it was shown that TNF-alpha maintains viability of neutrophils, thus allowing them to produce exaggerated inflammation responses. Scientists exposed neutrophils TNFalpha (100 ng/mL) in the presence or absence of antibodies to IL-8, and the extent of apoptosis was assessed. An enzyme-linked immunoassay was used to measure levels of the anti-apoptotic cytokine IL-8, induced by TNFalpha-stimulation. Because TNFalpha may mediate its effectthrough various cell-signaling pathways, the study next assessed the effect of kinase inhibition on the ability of TNFalpha to effect apoptosis and IL-8 production. Treatment with TNFalpha had a biphasic effect: at 4-8 h, apoptosis was increased but was markedly suppressed at 24 h (P<0.05). PMN cultured for 24 h with TNFalpha also showed markedly increased levels of IL-8. Neutralization of IL-8 inhibited the ability of TNFalpha to suppress apoptosis (P<0.05). These data illustrate a novel mechanism by which TNFalpha can indirectly elicit an anti-apoptotic effect via release of the anti-apoptotic chemokine IL-8 [76].

Perhaps one of the most tantalizing supporting evidences that TNF-alpha is a potential cause of ARDS are studies in which TNF-alpha was administered systemically as a cancer therapeutic and one of the adverse effects observed in some patients was a ARDS-type pathology [77].

Another cytokine which has been studied extensively in ARDS is interleukin-6. This cytokine is known topossess pro-inflammatory properties [78], as well as to suppress generation of T regulatory cells and promote Th17 cells [79-81]. It is accepted that in ARDS there is a reduction in T regulatory cells [82], whose role is tissue protection [83], and Th17 cells, which are commonly associated with inflammation [84]. In one study, 27 consecutive patients with severe medical ARDS. Plasma levels of tumor necrosis factor alpha (TNF-alpha) and interleukins (ILs) 1 beta, 2, 4, 6, and 8 were measured (enzyme-linked

immunosorbent assay [ELISA] method) on days 1, 2, 3, 5, 7, 10, and 12 of ARDS and every third day thereafter while patients were receiving mechanical ventilation. Subgroups of patients were identified based on outcome, cause of ARDS, presence or absence of sepsis, shock, and MODS at the time ARDS developed. Subgroups were compared for levels of plasma inflammatory cytokines on day 1 of ARDS and over time. Of the 27 patients, 13 survived ICU admission and 14 died (a mortality rate of 52%). Overall mortality was higher in patients with sepsis (86 vs 38%, p<0.02). The mean initial plasma levels of TNF-alpha, IL-1 beta, IL-6, and IL-8 were significantly higher in nonsurvivors (p<0.0001) and in those patients with sepsis (p<0.0001). Plasma levels of IL-1 beta (p<0.01) and IL-6 (p=0.03) were more strongly associated withpatient outcome than cause of ARDS (p=0.8), lung injury score (LIS), APACHE II score, sepsis (p=0.16), shock, or MODS score. Plasma levels of TNF-alpha, IL-1 beta, IL-6, and IL-8 remained significantly elevated over time (p<0.0001) in those who died. This study strongly supports the addition of IL-6 as another cytokine mediatory involved in the pathogenesis of ARDS [85].

A subsequent study examined 24 ARDS patients with MODS (ARDS+MODS group), 18 patients with ARDS but without MODS (ARDS group), and 55 patients with MODS but without ARDS as controls (control group). It was found that serum IL-6 levels in the ARDS+MODS group were significantly higher than those in the ARDS and MODS groups (P<0.01). The IL-6 levels increased with elevated ARDS illness severity (P<0.01); the sensitivity of IL-6 was high in all groups. Moreover, the IL-6 values were closely associated with patient survival [86]. Several other studies have shown correlation between IL-6 elevationand poor prognosis in ARDS [87-89].

Pterostilbene

Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) is a natural polyphenolic compound, primarily found in fruits, such as blueberries, grapes, and tree wood. It has been demonstrated to possess potent antioxidant and anti-inflammatory properties. It is a dimethylated analog of resveratrol which is found in blueberries [90], and is believed to be one of the active ingredients in ancient Indian Medicine [91]. The pterostilbene molecule is structurally similar to resveratrol, the antioxidant found in red wine that has comparable anti-inflammatory, and anticarcinogenic properties; however, pterostilbene exhibits increased bioavailability due to the presence of two methoxy groups which cause it to exhibit increased lipophilic and oral absorption [92-96]. In animal studies, pterostilbene was shown to have 80% bioavailability comparedto 20% for resveratrol making it potentially advantageous as a therapeutic agent [92].

We have demonstrated the pterostilbene administered in the form of nanostilbene in cancer patients results in increased NK cell activity, as well as interferon gamma production. Additionally, pterostilbene has shown to inhibit inflammatory cytokines associated with ARDS. For example, studies have demonstrated inhibition of interleukin-1 [97], interleukin-6 [98, 99], interleukin-8 [100], and TNF-alpha [101], by pterostilbene.

COVID-19 has been associated with endothelial activation and coagulopathy. It is interesting to note that numerous studies have demonstrated endothelial protective effects of pterostilbene. For example, Zhang et al. investigated the anti-apoptotic effects of pterostilbene in vitro and in vivo in mice. Exposure of human umbilical vein VECs (HUVECs) to oxLDL (200 μg/ml) induced cell shrinkage, chromatin condensation, nuclear fragmentation, and cell apoptosis, but pterostilbene protected against such injuries. In addition, PTinjection strongly decreased the number of TUNEL-positive cells in the endothelium of atherosclerotic plaque from apoE(−/−) mice. OxLDL increased reactive oxygen species (ROS) levels, NF-KB activation, p53 accumulation, apoptotic protein levels and caspases-9 and -3 activities and decreased mitochondrial membrane potential (MMP) and cytochrome c release in HUVECs. These alterations were attenuated by pretreatment. Pterostilbene inhibited the expression of lectin-like oxLDL receptor-1 (LOX-1) expression in vitro and in vivo. Cotreatment with PT and siRNA of LOX-1 synergistically reduced oxLDL-induced apoptosis in HUVECs. Overexpression of LOX-1 attenuated the protection by pterostilbene and suppressed the effects of pterostilbene on oxLDL-induced oxidative stress. Pterostilbene may protect HUVECs against oxLDL-induced apoptosis by downregulating LOX-1-mediated activation through a pathway involving oxidative stress, p53, mitochondria, cytochrome c and caspase protease [102]. Endothelial protection by pterostilbene [103, 104], and its analogue resveratrol are well known [105, 106].

Kalonji

First. Taking Kalonji increases the potency of the immune system [107, 108]. Specifically, it has been shown that kalonji activates the natural killer cells of the immune system. Natural killer cells, also calledNK cells are the body's first line of protection against viruses. It is well known that patients who have low levels of NK cells are very susceptible to viral infections. Kalonji has been demonstrated to increase NK cell activity. In a study published by Dr. Majdalawieh from the American University of Sharjah, Sharjah, United Arab Emirates [109], it was shown that the aqueous extract of Nigella sativa significantlyenhances NK cytotoxic activity. According to the authors, this supports the idea that NK cell activation by Kalonji can protect not only against viruses, but may also explain why some people report this herb has activity against cancer. It is known that NK cells kill virus infected cells but also kill cancer cells.

There are several publications that show that Kalonji has effects against cancer [110-124].

Second. Kalonji suppresses viruses from multiplying. If the virus manages to sneak past the immune system and enters the body, studies have shown that Kalonji, and its active ingredients such as thymoquinone, are able to directly stop viruses, such as coronaviruses and others from multiplying. For example, a study published from University of Gaziantep, in Turkey demonstrated that administration of Kalonji extract to cells infected with coronavirus resulted in suppression of coronavirus multiplication and reduction of pathological protein production [125]. Antiviral activity of Kalonji was demonstrated in other studies, for example, for example, viral hepatitis, and others [126].

Third. Kalonji protects the lungs from pathology. Kalonji was also reported by scholars to possess potent anti-inflammatory effects where its active ingredient thymoquinone suppressed effectively the lipopolysaccharide-induced inflammatory reactions and reduced significantly the concentration of nitric oxide, a marker of inflammation [127]. Moreover, Kalonji has been proven to suppress the pathological processes through blocking the activities of IL-1, IL-6, nuclear factor-κB [128], IL-1 β, cyclooxygenase-1, prostaglandin-E2, prostaglandin-D2 [129], cyclocoxygenase-2, and TNF-α [130] that act as potent inflammatory mediators and were reported to play a major role in the pathogenesis of Coronavirus infection.

Fourth. Kalonji protects against sepsis/too much inflammation. In peer reviewed study from King Saud University, Riyadh, Saudi Arabia, scientists examined two sets of mice (n=12 per group), with parallel control groups, were acutely treated with thymoquinone (ingredient from Kalonji) intraperitoneal injections of 1.0 and 2.0 mg/kg body weight, and were subsequently challenged with endotoxin Gram-negative bacteria (LPS O111:B4). In another set of experiments, thymoquinone was administered at doses of 0.75 and 1.0 mg/kg/day for three consecutive days prior to sepsis induction with live Escherichia coli. Survival of various groups was computed, and renal, hepatic and sepsis markers were quantified. Thymoquinone reduced mortality by 80-90% and improved both renal and hepatic biomarker profiles. The concentrationsof IL-1α with 0.75 mg/kg thymoquinone dose was 310.8±70.93 and 428.3±71.32 pg/ml in the 1 mg/kg group as opposed to controls (1187.0±278.64 pg/ml; P<0.05). Likewise, IL-10 levels decreased significantly with 0.75 mg/kg thymoquinone treatment compared to controls (2885.0±553.98 vs. 5505.2

±333.96 pg/ml; P<0.01). Mice treated with thymoquinone also exhibited relatively lower levels of TNF-α and IL-2 (P values=0.1817 and 0.0851, respectively). This study gives strength to the potential clinical relevance of thymoquinone in sepsis-related morbidity and mortality reduction and suggests that human studies should be performed [131].

Sulforaphane

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane], an isothiocyanate, is a chemopreventive photochemical which is a potent inducer of phase II enzyme involved in the detoxification of xenobiotics [132]. Sulforaphane is produced from the hydrolysis of glucoraphanin, the most abundant glucosinolate found in broccoli, and also present in other Brassicaceae [133]. Numerous studies have reported prevention of cancer [134-138], as well as cancer inhibitory properties of sulforaphane [139-144]. Importantly, this led to studies which demonstrated anti-inflammatory effects of this compound.

One of the fundamental features of inflammation is production of TNF-alpha from monocytic lineage cells.Numerous studies have shown that sulforaphane is capable of suppressing this fundamental initiator of inflammation, in part through blocking NF-kappa B translocation. For example, Lin et al. compared the anti-inflammatory effect of sulforaphane on LPS-stimulated inflammation in primary peritoneal macrophages derived from Nrf2 (+/+) and Nrf2 (−/−) mice. Pretreatment with sulforaphane in Nrf2 (+/+) primary peritoneal macrophages potently inhibited LPS-stimulated mRNA expression, protein expression and production of TNF-alpha, IL-1beta, COX-2 and iNOS. HO-1 expression was significantly augmentedin LPS-stimulated Nrf2 (+/+) primary peritoneal macrophages by sulforaphane. Interestingly, the anti-inflammatory effect was attenuated in Nrf2 (−/−) primary peritoneal macrophages. We concluded that SFNexerts its anti-inflammatory activity mainly via activation of Nrf2 in mouse peritoneal macrophages [145]. In a similar study, LPS-challenged macrophages were observed for cytokine production with or without sulforaphane pretreatment. Macrophages were pre-incubated for 6 h with a wide range of concentrations of SFN (0 to 50 μM), and then treated with LPS for 24 h. Nitric oxide (NO) concentration and gene expression of different inflammatory mediators, i.e., interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β, were measured. sulforaphane neither directly reacted with cytokines, nor with NO. To understand the mechanisms, the authors performed analyses of the expression of regulatory enzyme inducible nitic oxide synthase (iNOS), the transcription factor NF-E2-related factor 2 (Nrf2), and its enzyme heme-oxygenase (HO)-1. The results revealed that LPS increased significantly the expression of inflammatory cytokines and concentration of NO in non-treated cells. sulforaphane was able to prevent the expression of NO and cytokines through regulating inflammatory enzyme iNOS and activation of Nrf2/HO-1 signal transduction pathway [146]. These data are significant because studies have shown both TNF-alpha but also interleukin-6 are involved in pathology of COVID-19 [46, 47, 147-155]. The utilization of sulforaphane as a substitutefor anti-IL-6 antibodies would be more economical and potentially without associated toxicity. Other studies have also demonstrated ability of sulforaphane to suppress IL-6 [156-158]. Interestingly, a clinicalstudy was performed in 40 healthy overweight subjects (ClinicalTrials.gov ID NCT 03390855). Treatment phase consisted on the consumption of broccoli sprouts (30 g/day) during 10 weeks and the follow-up phase of 10 weeks of normal diet without consumption of these broccoli sprouts. Anthropometric parameters as body fat mass, body weight, and BMI were determined Inflammation status was assessed by measuring levels of TNF-α, IL-6, IL-1β and C-reactive protein. IL-6 levels significantly decreased (mean values from

4.76 pg/mL to 2.11 pg/mL with 70 days of broccoli consumption, p<0.001) and during control phase the inflammatory levels were maintained at low grade (mean values from 1.20 pg/mL to 2.66 pg/mL, p<0.001). C-reactive protein significantly decreased as well [159].

An additional potential benefit of sulforaphane is its ability to protect lungs against damage. It is known that the major cause of lethality associated with COVID-19 is acute respiratory distress syndrome (ARDS). It was demonstrated that sulforaphane is effective in the endotoxin model of this condition. In one experiments, BALB/c mice were treated with sulforaphane (50 mg/kg) and 3 days later, ARDS was inducedby the administration of LPS (5 mg/kg). The results revealed that sulforaphane significantly decreased lactate dehydrogenase (LDH) activity (as shown by LDH assay), the wet-to-dry ratio of the lungs and the serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (measured by ELISA), as well as nuclear factor-κB protein expression in mice with LPS-induced ARDS. Moreover, treatment with sulforaphane significantly inhibited prostaglandin E2 (PGE2) production, and cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9) protein expression (as shown by western blot analysis), as well as inducible nitric oxide synthase (iNOS) activity in mice with LPS-induced ALI. Lastly, the researchers reported pre-treatment with sulforaphane activated the nuclear factor-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway in the mice with LPS-induced ARDS [160].

Epigallocatechin-3-gallate (EGCG)

EGCG is similar to sulforaphane in that it has been reported to possess cancer preventative properties. This compound has been shown to be one of the top therapeutic ingredients in green tea. It is known from epidemiologic studies that green tea consumption associates with chemoprotective effects against cancer [161-171]. In addition, similarly to sulforaphane, EGCG has been shown to inhibit inflammatory mediators.

The first suggestion of this were studies shown suppression of the pro-inflammatory transcription factor NF-kappa B. In a detailed molecular study, EGCG, a potent antitumor agent with anti-inflammatory and antioxidant properties was shown to inhibit nitric oxide (NO) generation as a marker of activated macrophages. Inhibition of NO production was observed when cells were cotreated with EGCG and LPS. iNOS activity in soluble extracts of lipopolysaccharide-activated macrophages treated with EGCG (5 and 10 microM) for 6-24 hr was significantly lower than that in macrophages without EGCG treatment. Western blot, reverse transcription-polymerase chain reaction, and Northern blot analyses demonstrated that significantly reduced 130-kDa protein and 4.5-kb mRNA levels of iNOS were expressed inlipopolysaccharide-activated macrophages with EGCG compared with those without EGCG. Electrophoretic mobility shift assay indicated that EGCG blocked the activation of nuclear factor-kappaB, a transcription factor necessary for iNOS induction. EGCG also blocked disappearance of inhibitor kappaB from cytosolic fraction. These results suggest that EGCG decreases the activity and protein levels of iNOS by reducing the expression of iNOS mRNA and the reduction could occur through prevention of the binding of nuclear factor-kappaB to the iNOS promoter [172]. Another study supporting ability of EGCG to suppress NF-kappa B examined a model of atherosclerosis in which exposure of macrophage foam cells to TNF-α results in a downregulation of ABCA1 and a decrease in cholesterol efflux to apoA1, which is attenuated by pretreatment with EGCG. Moreover, rather than activating the Liver X receptor (LXR) pathway, inhibition of the TNF-α-induced nuclear factor-κB (NF-κB) activity is detected with EGCG treatment in cells. In order to inhibit the NF-κB activity, EGCG can promote the dissociation of the nuclear factor E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) complex; when the released Nrf2 translocates to the nucleus and activates the transcription of genes containing an ARE element inhibition of NF-κB occurs and Keap1 is separated from the complex to directly interact with IKKβ and thus represses NF-κB function [173].

The anti-inflammatory effects of EGCG can be seen in the ability of this compound to potently inhibit IL-6, the COVID-19 associated cytokine, in a variety of inflammatory settings. For example, in a cardiac infarct model, rats were subjected to myocardial ischemia (30 min) and reperfusion (up to 2 h). Rats were treated with EGCG (10 mg/kg intravenously) or with vehicle at the end of the ischemia period followed by a continuous infusion (EGCG 10 mg/kg/h) during the reperfusion period. In vehicle-treated rats, extensivemyocardial injury was associated with tissue neutrophil infiltration as evaluated by myeloperoxidase activity, and elevated levels of plasma creatine phosphokinase. Vehicle-treated rats also demonstrated increased plasma levels of interleukin-6. These events were associated with cytosol degradation of inhibitor kappaB-alpha, activation of IkappaB kinase, phosphorylation of c-Jun, and subsequent activation of nuclear factor-kappaB and activator protein-1 in the infarcted heart. In vivo treatment with EGCG reduced myocardial damage and myeloperoxidase activity. Plasma IL-6 and creatine phosphokinase levels were decreased after EGCG administration. This beneficial effect of EGCG was associated with reduction of nuclear factor-kB and activator protein-1 DNA binding [174]. In an inflammatory model of ulcerative colitis (UC) mice were randomly divided into four groups: Normal control, model (MD), 50 mg/kg/day EGCG treatment and 100 mg/kg/day EGCG treatment. The daily disease activity index (DAI) of the mice was recorded, changes in the organizational structure of the colon were observed and the spleen index (SI) was measured. In addition, levels of interleukin (IL)-6, IL-10, IL-17 and transforming growth factor (TGF)-β1 in the plasma and hypoxia-inducible factor (HIF)-1α and signal transducer and activator of transcription (STAT) 3 protein expression in colon tissues were evaluated. Compared with the MD group, the mice in the two EGCG treatment groups exhibited decreased DAIs and SIs and an attenuation in the colonic tissueerosion. EGCG could reduce the release of IL-6 and IL-17 and regulate the mouse splenic regulatory T-cell (Treg)/T helper 17 cell (Th17) ratio, while increasing the plasma levels of IL-10 and TGF-β1 and decreasing the HIF-1α and STAT3 protein expression in the colon. The experiments confirmed that EGCG treated mice with experimental colitis by inhibiting the release of IL-6 and regulating the body Treg/Th17 balance [175].

In patients with COVID-19, the ARDS associated with fatality resembles septic shock in many aspects, including DIC, fever, vascular leakage, and systemic inflammation. Wheeler et al. induced polymicrobialsepsis in male Sprague-Dawley rats (hemodynamic study) and C57BL6 mice (mortality study) via cecal ligation and double puncture (CL2P). Rodents were treated with either EGCG (10 mg/kg intraperitoneally) or vehicle at 1 and 6 h after CL2P and every 12 h thereafter. In the hemodynamic study, mean arterial blood pressure was monitored for 18 h, and rats were killed at 3, 6, and 18 h after CL2P. In the mortality study, survival was monitored for 72 h after CL2P in mice. In vehicle-treated rodents, CL2P was associated withprofound hypotension and greater than 80% mortality rate. Epigallocatechin-3-gallate treatment significantly improved both the hypotension and survival [176].

A subsequent study by Li et al. showed intraperitoneal administration of EGCG protected mice against lethal endotoxemia, and rescued mice from lethal sepsis even when the first dose was given 24 hours aftercecal ligation and puncture. The therapeutic effects were partly attributable to: 1) attenuation of systemic accumulation of proinflammatory mediator (e.g., HMGB1) and surrogate marker (e.g., IL-6 and KC) of lethal sepsis; and 2) suppression of HMGB1-mediated inflammatory responses by preventing clustering of exogenous HMGB1 on macrophage cell surface [177].

Finally, in a lung study mice were treated with EGCG (10 mg/kg) intraperitoneally (ip) 1 h before LPS injection (10 mg/kg, ip). The results showed that EGCG attenuated LPS-induced ARDS as it decreased the changes in blood gases and reduced the histological lesions, wet-to-dry weight ratios, and myeloperoxidase (MPO) activity. In addition, EGCG significantly decreased the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the lung, serum, and bronchoalveolar lavage fluid, and alleviated the expression of TLR-4, MyD88, TRIF, and p-p65 in the lung tissue. In addition, it increased the expression of IκB-α and had no influence on the expression of p65. Collectively, these results demonstrated the protective effects of EGCG against LPS-induced ARDS in mice through its anti-inflammatory effect that may be attributed to the suppression of the activation of TLR 4-dependent NF-κB signaling pathways [178].

Conclusion

QuadraMune represents an optimized nutraceutical blend formulated for inhibiting viral entry through stimulation of NK cells, suppressing viral proliferation through modulating the viral life cyclic, and dampening pathological inflammatory responses in order to allow the lungs sufficient protection from the ongoing cytokine storm. We anticipate to initiate clinical trials both in healthy volunteers at high risk of infection, and for infected patients.

EXAMPLES

Bleomycin (BLM) was administered intratracheally at 3 mg/kg in female 10 week old C57/BL6 mice. Mice were administered QuadraMune™ Combination by Gavage and/or Metformin daily after BLM challenge. Animals were sacrificed at day 12 and assessed for fibrosis (hydroxyproline content) and for cytokines. Results are shown in FIGS. 1-5.

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1. A method of inhibiting infection with SARS-CoV-2, suppressing proliferation of SARS-CoV-2 and inducing resolution of SARS-CoV-2 comprising administration of a therapeutic combination comprising: a) Green Tea and/or extract thereof; b) Blueberry and/or extract thereof; c) Nigella Sativa and/or extract thereof; d) broccoli and/or extract thereof and e) metformin
 2. The method of claim 1, wherein said green tea extract is epigallocatechin-3-gallate or an analogue thereof.
 3. The method of claim 1, wherein said blueberry extract is pterostilebene or an analogue thereof.
 4. The method of claim 1, wherein said Nigella Sativa extract is thymoquinone or an analogue thereof.
 5. The method of claim 1, wherein said broccoli extract is sulforaphane or an analogue thereof.
 6. The method of claim 1, wherein said therapeutic combination is administered at a dosage and frequency sufficient to inhibit viral establishment into the host.
 7. The method of claim 6, wherein inhibition of viral establishment into the host is accomplished by enhancement of natural killer cell activity.
 8. The method of claim 6, wherein inhibition of viral establishment into the host is accomplished by enhancement of interferon production.
 9. The method of claim 1, wherein said therapeutic combination is administered at a dosage and frequency sufficient to inhibit viral replication into the host.
 10. The method of claim 9, wherein said viral replication in the host is associated with suppression of the viral life cycle.
 11. The method of claim 1, wherein said therapeutic mixture decreases propensity towards acute respiratory distress syndrome (ARDS).
 12. The method of claim 11, wherein said ARDS is associated with activation of complement.
 13. The method of claim 11, wherein said ARDS is associated with enhanced expression of adhesion molecules on endothelial cells.
 14. The method of claim 13, wherein said adhesion molecules are selected from a group comprising of: a) E selectin; b) ICAM-1; c) VLA-4 and; d) Cadherin.
 15. The method of claim 14, wherein said ARDS is associated with enhanced monocytic accumulation in the alveolar space.
 16. The method of claim 11, wherein said ARDS is associated with enhanced neutrophil accumulation in the alveolar space.
 17. The method of claim 16, wherein said neutrophils are activated.
 18. The method of claim 17, wherein said activated neutrophils produce matrix metalloproteases.
 19. The method of claim 1, wherein said therapeutic composition reduces expression of inflammatory markers.
 20. The method of claim 19, wherein said inflammatory markers are selected from the group consisting of: Cluster of differentiation 40 ligand (CD-40L), Eotaxin, fibrinogen, growth hormone (GH), keratinocyte-derived cytokine (KC/GRO), interleukin-1.beta. (IL-1.beta.), IL-6, IL-18, lymphotactin, myeloperoxidase (MPO), tissue inhibitor of metalloproteinase 1 (TIMP-1), C-reactive protein (CRP), macrophage-derived chemokine (MDC), macrophage inflammatory protein-1.alpha. (MIP-1.alpha.), vWF, and oncostatin 